Limits...
Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

Show MeSH
Comparison of depletion of various proteins in TetO7 and Tet07-Ubi-Leu strains. Growth curves (left) and Western blot analysis (middle and right) of strains depleted of essential proteins Dbp4p (A), Bfr2p (B) Erg1 (C), Sec62 (D), Prp19 (E), and Rrn3 (F) using different depletion cassettes. Samples were collected at the indicated time points following the addition of doxycycline. The individual proteins were visualized using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is shown on the right; a mean of two independent experiments is shown. For growth curves (left charts), the mean of three independent experiments is plotted; vertical y-axes are in logarithmic scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4325845&req=5

Figure 4: Comparison of depletion of various proteins in TetO7 and Tet07-Ubi-Leu strains. Growth curves (left) and Western blot analysis (middle and right) of strains depleted of essential proteins Dbp4p (A), Bfr2p (B) Erg1 (C), Sec62 (D), Prp19 (E), and Rrn3 (F) using different depletion cassettes. Samples were collected at the indicated time points following the addition of doxycycline. The individual proteins were visualized using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is shown on the right; a mean of two independent experiments is shown. For growth curves (left charts), the mean of three independent experiments is plotted; vertical y-axes are in logarithmic scale.

Mentions: To determine whether the TetO7-Ubi-DAA cassettes can be used in general to reduce depletion times, we tested the strategy with six other essential proteins. We selected proteins from different cellular processes and spanning three orders of magnitude in their cellular abundance (Table 1). Depletion of all these proteins in strains carrying the TetO7-Ubi-Leu cassette was significantly faster than in strains with only the TetO7 promoter (Figure 4). In all cases, the depletion time required for an obvious deviation of growth of the depleted versus nondepleted control strains was several hours shorter. The individual protein depletion was also faster in the TetO7-Ubi-Leu strains. The Western blotting analyses revealed that the steady-state levels of Bfr2p, Dbp4p, Prp19, and Rrn3 were reduced already in the absence of doxycycline in TetO7-Ubi-Leu strains, as could be expected, due to the faster turnover of the Ubi-Leu fusion protein. Interestingly, only the TetO7-Ubi-Leu-Dbp4 strain exhibited slightly slower growth in the absence of doxycycline, whereas the other strains were not affected. It is likely that the reduced steady-state levels of Dbp4p in the TetO7-Ubi-Leu strain are already limiting for growth and therefore, upon the start of depletion, the cell growth diminishes more or less immediately, as evidenced by the growth curve (Figure 4A). In this case, use of a less-destabilizing DAA, such as isoleucine, can be used to increase the steady-state (nondepleted) levels of the target protein.


Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Comparison of depletion of various proteins in TetO7 and Tet07-Ubi-Leu strains. Growth curves (left) and Western blot analysis (middle and right) of strains depleted of essential proteins Dbp4p (A), Bfr2p (B) Erg1 (C), Sec62 (D), Prp19 (E), and Rrn3 (F) using different depletion cassettes. Samples were collected at the indicated time points following the addition of doxycycline. The individual proteins were visualized using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is shown on the right; a mean of two independent experiments is shown. For growth curves (left charts), the mean of three independent experiments is plotted; vertical y-axes are in logarithmic scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325845&req=5

Figure 4: Comparison of depletion of various proteins in TetO7 and Tet07-Ubi-Leu strains. Growth curves (left) and Western blot analysis (middle and right) of strains depleted of essential proteins Dbp4p (A), Bfr2p (B) Erg1 (C), Sec62 (D), Prp19 (E), and Rrn3 (F) using different depletion cassettes. Samples were collected at the indicated time points following the addition of doxycycline. The individual proteins were visualized using antibodies against the HA tag and actin. The quantification of the blots, normalized to actin levels, is shown on the right; a mean of two independent experiments is shown. For growth curves (left charts), the mean of three independent experiments is plotted; vertical y-axes are in logarithmic scale.
Mentions: To determine whether the TetO7-Ubi-DAA cassettes can be used in general to reduce depletion times, we tested the strategy with six other essential proteins. We selected proteins from different cellular processes and spanning three orders of magnitude in their cellular abundance (Table 1). Depletion of all these proteins in strains carrying the TetO7-Ubi-Leu cassette was significantly faster than in strains with only the TetO7 promoter (Figure 4). In all cases, the depletion time required for an obvious deviation of growth of the depleted versus nondepleted control strains was several hours shorter. The individual protein depletion was also faster in the TetO7-Ubi-Leu strains. The Western blotting analyses revealed that the steady-state levels of Bfr2p, Dbp4p, Prp19, and Rrn3 were reduced already in the absence of doxycycline in TetO7-Ubi-Leu strains, as could be expected, due to the faster turnover of the Ubi-Leu fusion protein. Interestingly, only the TetO7-Ubi-Leu-Dbp4 strain exhibited slightly slower growth in the absence of doxycycline, whereas the other strains were not affected. It is likely that the reduced steady-state levels of Dbp4p in the TetO7-Ubi-Leu strain are already limiting for growth and therefore, upon the start of depletion, the cell growth diminishes more or less immediately, as evidenced by the growth curve (Figure 4A). In this case, use of a less-destabilizing DAA, such as isoleucine, can be used to increase the steady-state (nondepleted) levels of the target protein.

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

Show MeSH